Figure 2

Activated phenotype of mouse dermal fibroblast (mDF) constitutively expressing Ha-Ras. (A) Left panel, pSmad3, Smad3, pERK1/2, ERK1/2, Ha-Ras and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) immunoblots of protein extracts from mDF cultures infected with Ha-Ras (Ha-Ras lenti) or control (lenti) lentiviruses at 6 h post-infection (n = 3 per each sample) with the bar graphs on the side summarizing the relative ratios of pSmad3 over GADPH and pERK1/2 over ERK1/2; right panel, luciferase activity of a Smad3-responsive plasmid transiently transfected in mDF infected with Ha-Ras or control lentivirus and/or stimulated with recombinant transforming growth factor-β (TGFβ1; 2 ng/mL) for 6 h. (B) Collagen I and Ha-Ras immunoblots of protein extracts from mDF cultures infected with Ha-Ras or control lentiviruses at 24 h post-infection and in control cells treated with recombinant TGFβ1 (2 ng/mL) for the same length of time (n = 3 per each sample). (C) α-smooth muscle actin (αSMA) immunostaining of mDF infected with Ha-Ras or control lentiviruses with bar graphs on the side summarizing the percentage of αSMA-positive cells 24 h after the infection; measurements were performed on >100 cells from three independent infections. (D) pSmad3 immunoblots of protein extracts from mDF cultures treated with the MEK inhibitor PD98059 (10 μM) for 2 h prior to infection for 6 h with Ha-Ras or control lentiviruses with bar graphs on the side summarizing the relative ratio of pSmad3 and the loading control GADPH in the various experimental samples (n = 3 per each sample). (E) Collagen I immunoblots of protein extracts from mDF cultures treated with the MEK inhibitor PD98059 (10 μM) for 2 h prior to infection with Ha-Ras or control lentiviruses (n = 3 per each sample); protein levels were assessed 24 h post-infection and the bar graphs on the side summarize the relative ratio of collagen I and the loading control GADPH in the various experimental samples. (F) Luciferase activity (expressed as fold induction over control sample) of the COL1A2 promoter plasmid transiently transfected in primary mDF infected with Ha-Ras or control lentiviruses at 24 h post-infection with or without 2 h pre-treatment with the MEK inhibitor PD98059 (10 μM). In the relevant panels, arrows and arrowheads point to Ha-Ras and a non-specific immunoreactive products or pSmad3 and an unidentified cross-reacting R-Smad, respectively; luciferase values represent the average of three independent transfections each performed in duplicate; error bars signify ± standard deviation and asterisks indicate statistically significant differences (P < 0.05).